الفهرس 
Molecular characteristics and Life cycle of HCVOnly 14 pages are availabe for public view
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Abstract
Hepatitis C virus (HCV) is one of the most important pathogens of the human and is able to cause mild to severe liver diseases and a potential cause of substantial morbidity and mortality worldwide. The most estimates of disease burden show an increase in seroprevalence over the last 15 years to 2.8%, equating to >185 million infections worldwide. Based on the general trends for most other diseases, 75% of HCV-infected individuals developing chronic liver disease. Of those HCV-infected patients who develop chronic liver disease 1.6% progress to hepatocellular carcinoma (HCC), a condition with a mortality rate >80%. Although, the mechanism of HCV infection outcomes is not well defined, it is believed that immunological mechanisms such as cytokine production are involved in HCV pathogenesis.
Interferon-γ (IFN-γ) is a multifunctional cytokine produced mainly by NK cells and activated T cells that plays a critical role in host immune responses against pathogens and cancer. The bio-activity of IFN-γ has been studied extensively in human the major producers of this gene are activated T-cells, natural killer (NK) and NKT-cells. This gene has been demonstrated to play a key role in pathogen clearance and tumor surveillance.
Although the antiviral effect of IFN-alpha on the replication of HCV has been in the focus of research, surprisingly little is known about the role of other cytokines such as (IFN-γ) in the cellular defense against HCV.
It seems that multiple arms of the immune response, mainly cellular immunity, play an important role in the immunopathogenesis of HCV infection. The type of early immune response that the infected host is able to mount following infection is believed to dictate strongly the outcome toward resolution or persistence of HCV infection.
The reasons for the lack of responsiveness and the mechanisms that contribute to the failure of innate and adaptive immune responses to eliminate viral infection in chronically infected patients are not well understood.
The aim of the present study was to understand the role of (IFN-γ) which could play in HCV immunopathogensis, follow-up of it before, during and after theraby. Also, studying the roles of CD4, CD3 and natural killer cells by measurment their percentages before, during and after theraby.
Serum samples have been collected and classified into:
1) 110 chronic hepatitis C patients (CHC).
2) 30 hepatocellular carcinoma patients (HCC) infected with HCV.
3) 30 healthy individuals.
Patient sera have been kindly supplied from El Demerdash and Ain shams University Hospitals, Cairo, Egypt. The clinical status of patients recorded concerning age, gender, biochemical measurements (liver enzymes).
Many techniques have been used in this study:
1) Detection of interferon-γ using ELISA, SDS-PAGE and Western blot techniques.
2) Measurement of CD4, CD3, and NK percentage in whole blood using flow cytometer.
Data had been analyzed using statistical software package (SPSS) for Microsoft windows, SPSS Inc.)
We can summarize our results in the following points:
1) Mean of age in HCC group, CHC group and healthy Control group was 50.2±11.6, 47.5±14.1 and 48.51±4.9, respectively. P value > 0.05 which means that there is no significance differences between tested groups respecting their age. On the other hand, the percentage of male to female in HCC group was 56.7%/43.3% respectively. In CHC it was 57.3% /42.7% respectively. In Control group it was 53.3%/46.7% respectively. No significance differences was obtained among tested groups (P value > 0.05).
2) Highly significance differences was shown among tested groups regarding ALT, AST, total bilirubin, direct bilirubin and albumin liver enzymes. The mean of ALT was 72.7±3.4 IU/L, 67.4±4.5 IU/L and 28.0±5.2 IU/L in HCC, CHC and Control groups, respectively (P value < 0.01). Also, the mean of AST was 75.3±1.6 IU/L, 52.1±5.7 IU/L and 20.1±1.3 IU/L in the above groups, respectively (P value < 0.0001). The mean of total bilirubin was 2.20±0.12 mg/dL, 1.5±0.2, 0.70±0.10 mg/dL in the above groups, respectively (P value < 0.001). The mean of direct bilirubin was 1.10±0.05 mg/dL, 0.23±0.02, 0.16±0.01 mg/dL in the above groups, respectively (P value < 0.0001). The level of albumin on the other hand, was also highly significance among tested groups showing 1.80±0.09 g/dL, 2.90±0.10 g/dL and 5.40±0.06 g/dL (P value < 0.0001) in HCC, CHC and Control groups.
3) Serum samples from HCC infected with HCV, chronic hepatitis C patients and healthy individuals were analyzed by 12 % one-dimensional SDS-PAGE under reducing conditions and staining with Coomassie blue. The Coomassie blue stained separated polypeptides. By using Western blot technique, intense sharp bands (26 kDa) were shown in samples from HCC patients infected with HCV as well as in those chronically hepatitis C patients. No reaction was detected in serum samples from healthy individuals.
4) The cut-off level of ELISA above or below the calculated value (OD=0.30 that equal 37 µg/ml) was considered to determine the positivity or negativity of tested samples.
5) Extremely high significant difference (P <0.0001) was shown among tested groups regarding IFN-γ level. The mean ± standard error of mean (SEM) of serum IFN-γ level was 53.0±2.1 µg/ml, 44.2±0.8 µg/ml, 23.5±0.7 µg/ml in HCC, CHC and Control groups, respectively. The differences were also remain statistically significant (P <0.0001) when CHC group was compared with HCC group.
6) Extremely high significant difference (P <0.0001) was shown among tested groups regarding IFN-γ level. The mean ± standard error of mean (SEM) of serum IFN-γ level was 53.0±2.1 µg/ml, 50.0±2.3 µg/ml, 42.1±0.6 µg/ml in HCC, in CHC with non-cirrhotic liver and in CHC with cirrhotic liver, respectively. There was also statistically significant (P < 0.05) when CHC patients with cirrhotic liver was compared with HCC group.
7) Extremely high significant difference (P <0.0001) was shown among tested groups regarding IFN-γ level. The mean ± standard error of mean (SEM) of serum IFN-γ level was, increase with progression of liver fibrosis where it was 38.6±1.2 µg/ml, 43.7±0.6 µg/ml and 50.0±2.3 µg/ml in mild (F0-F1), moderate (F2-F3), and advanced (F4-F6) fibrosis stages, respectively.
8) Peripheral blood samples of CHC and HCC patients in addition to healthy individuals were collected and the different lymphocyte populations, were analyzed by flow cytometry to measure CD4, CD3, and CD3-/CD16+CD56+ NK cells.
9) The frequency of CD3+ T-cells, CD4+ Th-cells CD3-/CD16+ and CD3- /CD56+ NK cells was significantly low in HCC patients than those of CHC or healthy individuals, low in HCC patients than those of Non-cirrhotic or Cirrhotic, low in advanced patients (F4-F6) than those of Mild (F0-F1) or Moderate (F2-F3), low in Decompensated patients than those of compensated individuals (p < 0.05 to P < 0.01 to P < 0.0001 for all comparisons).
10) After following up 70 CHC patients classified into 40 non-cirrhotic and 30 cirrhotic (15 compensated and 15 decompensated patients), liver function parameters including ALT, AST, bilirubin and albumin all improved after antiviral therapy. ALT and AST levels normalized and showed an extremely significant decrease (P= 0.0003 and 0.0002; respectively). Also, bilirubin level significantly improved after the antiviral therapy. Subsequently, albumin levels also significantly improved (P < 0.0001).
11) After 3 months and 6 months of daclatasvir and sofosbuvir combination treatment for non-cirrhotic and compensated groups., and daclatasvir, sofosbuvir and ribavirin combination treatment for decompensated group, serum concentration of IFN-γ was found to be significantly reduced in all studied groups.
12) The frequency of CD3+ T-cells, CD4+ Th-cells, CD3-CD16+, and CD3- CD56+ NK cells was significantly increased in non-cirrhotic CHC, compensated cirrhotic CHC and decompensated cirrhotic CHC patients at 6 months after combination therapy (P < 0.5 to P < 0.001to p < 0.0001; respectively).
According to our results, interferon gamma (IFN-γ) and the studied CD3+ T-cells, CD4+ Th-cells, CD3-CD16+, and CD3- CD56+ NK cells have a potential role in the evaluation of HCV infected patients prognosis and could be used as an immunological indicators for the response of treatment.
Our future hope in a future study to focus on studying interferon gamma in responders compared to non-responders as our results may support the hypothesis that pretreatment may affect gamma interferon gene expression level in PBMCs and its protein could be used to predict treatment outcome. As serum interferon gamma may be higher in PBMCs of non-responders when compared to responders but this waits further confirmatory study. Also, studying the host immune response in both groups to confirm our results.